Results from omic approaches in rat or mouse models exposed to inhaled crystalline silica: a systematic review

Background Crystalline silica (cSiO2) is a mineral found in rocks; workers from the construction or denim industries are particularly exposed to cSiO2 through inhalation. cSiO2 inhalation increases the risk of silicosis and systemic autoimmune diseases. Inhaled cSiO2 microparticles can reach the alveoli where they induce inflammation, cell death, auto-immunity and fibrosis but the specific molecular pathways involved in these cSiO2 effects remain unclear. This systematic review aims to provide a comprehensive state of the art on omic approaches and exposure models used to study the effects of inhaled cSiO2 in mice and rats and to highlight key results from omic data in rodents also validated in human. Methods The protocol of systematic review follows PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. Eligible articles were identified in PubMed, Embase and Web of Science. The search strategy included original articles published after 1990 and written in English which included mouse or rat models exposed to cSiO2 and utilized omic approaches to identify pathways modulated by cSiO2. Data were extracted and quality assessment was based on the SYRCLE’s Risk of Bias tool for animal studies. Results Rats and male rodents were the more used models while female rodents and autoimmune prone models were less studied. Exposure of animals were both acute and chronic and the timing of outcome measurement through omics approaches were homogeneously distributed. Transcriptomic techniques were more commonly performed while proteomic, metabolomic and single-cell omic methods were less utilized. Immunity and inflammation were the main domains modified by cSiO2 exposure in lungs of mice and rats. Less than 20% of the results obtained in rodents were finally verified in humans. Conclusion Omic technics offer new insights on the effects of cSiO2 exposure in mice and rats although the majority of data still need to be validated in humans. Autoimmune prone model should be better characterised and systemic effects of cSiO2 need to be further studied to better understand cSiO2-induced autoimmunity. Single-cell omics should be performed to inform on pathological processes induced by cSiO2 exposure. Supplementary Information The online version contains supplementary material available at 10.1186/s12989-024-00573-x.


Background
Crystalline silica (cSiO 2 ) is a major component of rocks such as granite or sand which is found in materials used in the building industries such as cement or kitchen worktops.Workers from the construction or denim production industries as well as miners are particularly exposed to cSiO 2 through inhalation [1].Inhalation of cSiO 2 increases the risk of respiratory disorders such as silicosis, but also increases the risk of systemic autoimmune diseases such as Systemic Sclerosis (SSc), Systemic Lupus Erythematosus (SLE) or Rheumatoid Arthritis (RA) [2][3][4][5][6].
Inhaled cSiO 2 microparticles enter into the respiratory tract and can reach pulmonary alveoli [7].The phagocytosis of cSiO 2 particles by alveolar macrophages can induce cytotoxicity and macrophage cell death [8,9] and can also initiate inflammatory responses and fibrosis through NLRP3 inflammasome activation [10].Moreover, cSiO 2 -induced lung cell death is responsible for self-dsDNA release, STINGmediated sensing, IFN response and inflammation [11].This process can be favoured by the impairment of efferocytosis capacities of macrophages exposed to cSiO 2 [12].cSiO 2 is also known to induce systemic autoimmunity [13] but pathophysiological mechanisms involved in these effects remain unclear.
Omic methods are high-throughput technologies increasingly used in human and animal studies since the 1990s.They notably explore genomic, transcriptomic, proteomic or metabolomic data without a priori.They allow a better understanding of the overall biological processes and pathways involved in many disorders [14,15] or in response to xenobiotic exposure including pollutants such as diesel exhaust particles [16].Several studies using omic approaches have explored pathways involved in cSiO 2 toxicity in mouse and rat models, but a comprehensive overview of these results is still lacking.Access to biological samples from patients exposed to cSiO 2 is limited (limited access to bronchoalveolar lavages or lung biopsy) but rodent models can reflect cSiO 2 exposure in human.Therefore, mechanisms identified in rodent models could also be relevant for humans [17].A better identification of processes and pathways underlying cSiO 2 toxicity in mouse or rat models may help design new therapeutic targets for cSiO 2 -related diseases in humans.
This systematic literature review (SLR) aimed at 1) providing a comprehensive state of the art on exposure methods and omic approaches used to study the effects of inhaled cSiO 2 in mice and rats and 2) identify key results from omic data involved in cSiO 2-related disorders and highlight those validated in human.

Methods and analysis
The report for this SLR was designed in accordance with the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines [18].This protocol was registered in February 2022 on the International prospective register of systematic reviews Prospero (ID n°CRD42022299944) before research started.

Search strategy
The search strategy aimed at selecting original studies mentioning cSiO 2 , mouse or rat models and at least one omic method, as summarised in the PECO form (see Additional file 1).
PubMed (Medline), Embase and Web of Science were selected for the identification of eligible titles and abstracts published before January 3, 2022.A specific search term strategy was designed for each database (see Additional file 2).Search equations were designed with the help of librarians.To test the relevance of our search strategies, eleven milestone articles considered as mandatory for our research question were selected a priori, based our knowledge from the field [19][20][21][22][23][24][25][26][27][28][29].All eleven milestone articles were retrieved in each database confirming the relevance of the selected search terms.The first publications using omic techniques was published in the 90's, therefore only articles published after 1990 were explored.As this SLR focused on cSiO 2 , titles and abstracts mentioning nanoparticles of silica were excluded.Only rat or mouse experimentations were kept, others in vivo or in vitro models were excluded.Omic methods selected for this systematic review were genomics, transcriptomics, proteomics or metabolomics.Selected omic methods included RNAseq, iTRAQ, Nanostring, microarray or mass spectrometry at the bulk or single-cell levels.

Study selection
Title and abstract screening was performed using Rayyan software (https:// www.rayyan.ai/).Two reviewers (AL & LM) independently screened all titles and abstracts, after publication and validation of the SLR protocol on Prospero.There were two screening phases for article selection.The first one consisted in screening titles and abstracts and the second selection consisted in screening selected full texts to include only relevant manuscripts for data extraction.A third reviewer (VL) resolved disagreements between reviewers for abstract and full text screening.

Title and abstract screening
Title and abstract screening aimed at pre-selecting articles that included mouse or rat models, silica exposure and the use of at least one omic method.Studies mentioning silica gel or column, silica-coated beads, silica nanoparticles or micro/nanospheres, mesoporous silica, silica spicules or bleomycin exposure were excluded.Studies without available English abstract were also excluded.

Full text screening
Inclusion and exclusion criteria for final article selection are summarized in Table 1.Briefly, only studies on mouse or rat models exposed to cSiO 2 by inhalation were included.All strain, sex and age of mice and rats could be included except genetically modified animals such as knockout mice or rats because they were considered too different from the physiological conditions, and such results could not be applied to human physiology.NZBWF1 and NZM2410 were included because of their spontaneous genetic background for autoimmunity, similarly to what could be observed in human.All doses and frequency of exposure to cSiO 2 were included.Only studies using omic approaches were finally kept for data extraction.Outcomes based on omic techniques were kept, including -but not limited to-genomic, transcriptomic, proteomic or metabolomic analyses; gene or protein expression profiles; miRNA-expression profiling/levels; RNA-seq; NanoString nCounter; iTRAQ; single-cell; microarray analyses; SAGE-seq; LC-MS.Outcomes assessed through other technological approaches than omic methods were excluded.

Assessment of methodological quality
The quality of animal experiments was evaluated following the SYRCLE's Risk of Bias (RoB) tool for animal studies [30].This tool uses ten items to evaluate experimental bias and ten related questions.The response options are: "1 = yes" indicating that the study follows criteria of evaluation and it is free of bias; "0 = no" indicating the presence of bias in the study; "Un = unclear" indicating the absence of mention about these criteria in the study, with subsequent unclear risk of bias.The evaluation of quality assessment was performed by LM and checked by AL and VL.

Data extraction
Data extraction was performed by LM and checked by AL and VL following a data extraction template that was adapted throughout the process.This template included the following items: 1. article characteristics including title, author, publication year; 2. Animal model including species, strain, age, sex, number of animals per group, control group considered as reference; 3. cSiO 2 characteristics including size, purity and exposure including exposure route, dosage, frequency; 4. omic methods used, organ studied and outcomes including timing of measurement.Main biological processes, cellular components, molecular functions, pathways, networks and markers (gene, protein, miRNA) modulated by cSiO 2 were extracted.Data were expressed in the format provided in the articles in accordance with existing databases for pathways, biological processes, networks and markers (Gene Ontology (GO), Kyoto encyclopaedia

Rat and mouse characteristics
The characteristics of animal models used in included studies are described in Table 2. Rats were more frequently used than mice (N = 23 (56%)).The majority of Fig. 1 Process of inclusion and exclusion of studies animals was males (N = 30 (66.7%)) and their age at the beginning of experiment was comprised between 6 to 10 weeks (N = 25 (59.6%)).Some studies did not mention the sex (N = 2 (4.4%)) and the age (N = 1 (2.4%)) of animals.Few studies (N = 6 (14.6%)) used mouse strains prone for autoimmune diseases such as lupus-prone NZBWF1 and NZM2410.The number of animals per experimental group was commonly between 6 and 10 (N = 20 (46.5%)).However, the number of animals specifically studied with omic methods was lower than 5 in 38.6% (N = 17) of the studies and 31.8%(N = 14) of studies did not mention this number (N = 14 (31.8%)).

Crystalline silica exposure
Characteristics of cSiO 2 as well as the timing, dose and frequency of exposure are shown in Table 3. Half of the studies (N = 22 (53.6%))exposed mice or rats to Min-U-Sil-5 silica, measuring 1.5-2 µm with a purity higher than 99% [60].Other types of cSiO 2 with same size and purity were used in several studies such as the silica from Sigma Aldrich mainly used on rat models (N = 10 (24.4%)) and Forsman Scientific only used on mouse models (N = 3 (7.3%)).Only one study exposed rats to DQ12, a silica with average size of 2.2 µm and purity of  87% [60].Among the types of exposures, intratracheal instillation and inhalation chamber were the most chosen ones (N = 15 (36.6%) and N = 14 (34.1%)respectively), although inhalation chambers were only used on rats.
Among chronic exposure, the daily cSiO 2 exposure concerned only rat models.Twenty percent of the studies (N = 9) used chronic exposure on rats 6 h/day with dosage mainly comprised between 2 to 15 mg/m 3 (N = 9) during several days (minimum of 5 days) or weeks (maximum of 12 weeks).The timing of outcome measurement was mostly comprised between 2 and 11 weeks for mice (N = 16 (25%)) and higher than 12 weeks for rats (N = 14 (21.9%)).

Omic methods and organ studied
The summary of omic methods and organs or fluids used in studies is presented in additional file (see Additional file 4).The omic methods were more commonly applied to lung samples but blood, serum, plasma, kidney and spleen were also evaluated in some experiments.Sub-acute effects of cSiO 2 exposure were more represented through omic methods (N = 30) than acute effects (N = 10) or long-term effects (N = 24), considering that one single study could use multiple omic approaches at the same time.All types of omics are provided in Table 4.This table highlights the diversity of techniques, particularly in transcriptomic and proteomic analyses, which can limit comparability among studies.Transcriptomic studies represented 76% (N = 35) of all omic methods used whereas proteomics represented 19.6% (N = 9) and metabolomics 4.3% (N = 2).mRNA microarray and mRNA-sequencing were the most commonly used transcriptomics methods (34.8% (N = 16) and 10.9% (N = 5) respectively).Among proteomic studies, mass spectrometry (N = 5 (10,9%)) and protein microarray (N = 4 (8,7%)) were mainly used.
As biological function is carried by proteins, we checked whether a change in mRNA expression was also related to a change at the protein level (Table 5).The number of transcriptomic studies for which mRNA results were validated at protein level represented 39.3% (N = 11) and the main techniques used were western blotting (14.3%, N = 4), ELISA/multiplex assays (21.4%, N = 6) and immunohistology (17.9%,N = 5).

Effects of crystalline silica as assessed by omic approaches in mouse
Lung was the most studied organ in rat and mouse models (N = 21/23 and N = 17/18 respectively) (see Additional file 5).Outcomes studied through omic methods after cSiO 2 exposure at several time points in the lungs are shown in Fig. 2. Biological processes, pathways, networks and mRNA expression were the most frequently reported outcomes.
Heatmap representing the main domains of biological processes, pathways and networks found in omic approaches is presented in Fig. 3. Biological processes comprised mostly cellular and immunity domains while pathways and networks mostly included immunity and inflammation.The other domains were diseases, IFN response, transcription factor, ECM production, stress and cell death.Cellular response was the third most represented sub-domain in which movement, response to stimulus, adhesion, communication and growth/proliferation terms were mainly found (Additional file 6).Detailed characterisation of "Immunity" and "inflammation" in lungs or at systemic levels (serum, plasma, and spleen) is provided in Fig. 4A and B respectively.Several terms were common to the pulmonary and systemic levels such as those related to cytokines, notably IL-10, TNF, IL-6, IL-17 and IL-1 terms.In the lungs, several "immunity" and "inflammation" terms were linked to immune cell recruitment and activation such as cell activation, movement/adhesion, antigen presentation, aggregation, communication, cytokines, chemokines, B-cell, T-cell and macrophage functions."Complement" term retrieved in the lungs could be linked to "phagocytosis" as complement-induced phagocytosis is a major innate immune mechanism.At systemic level (i.e.mostly blood and serum) in context of long-term cSiO 2 exposure, adaptive immune response was frequently reported with the terms related to T-and B-cell function, antibody and antigen presentation.

Validation of omic data in humans
Omic results obtained from animal studies provide data on gene and protein expression during exposure to cSiO 2 , however, these results need to be confirmed in humans to demonstrate their relevance for patients.Among the 41 included studies, only 17% (N = 7) validated their results from rodent in human samples (Table 6).The majority of these studies validated their results in seric or lung samples from patients with silicosis (N = 4).However, three studies validated their data in patients with idiopathic pulmonary fibrosis (IPF) which was less relevant to demonstrate the impact of cSiO 2 exposure in human.
Pang et al. [22] and Shichino et al. [24] consistently showed changes in lipid metabolism in response to cSiO 2 exposure in mice and confirmed these results in the lungs of patients with silicosis and/or IPF (Table 6).The arachidonic acid (AA) pathway was found activated and prostaglandin D2 (PGD2) and thromboxane A2 (TXA2) were upregulated in cSiO 2 -exposed mice and silicosis human lungs [22].In addition, Srebf1, a transcription factor involved in the regulation of lipid metabolism, was downregulated in cSiO 2 -exposed mouse and the lungs of patients with IPF [24].Gremlin1, an antagonist of bone morphogenetic protein (BMP), was found upregulated independently both in cSiO 2 -exposed mice and in the lungs of patients with IPF [45,56], demonstrated the importance of this inhibitor as a key pro-fibrotic factor.
None of the omic studies exploring the systemic effects of cSiO 2 in rodents validated their results in humans.This lack of data identifies a gap in the literature, with a need to fully validate systemic effects of cSiO 2 and related systemic pathways.

Discussion
This review provides a comprehensive state of the art on exposure methods and omic approaches used to study the effects of inhaled cSiO 2 in mice and rats.Our work highlighted that male gender and rat models were more frequently used than females and mice and only a limited number of studies used mouse strains prone to autoimmunity, although it is a well endorsed effect of cSiO 2 in human.Both acute and chronic exposure were explored in rats and mice and acute, sub-acute and long-term effects being mainly observed in the lungs.Transcriptomic approaches were more commonly performed, only a few studies used proteomic, metabolomic or single-cell omic approaches.Biological processes, pathways and networks were the most commonly reported outcomes in omic analyses.Among these outcomes, immunity and inflammation were the top two domains that were reported as impacted by exposure to cSiO 2 in the lungs.Interestingly, only a few studies validate their transcriptomic results at the protein level in rodents and the number of translational studies was less than 20%, with only a few studies validating their results in humans.

Experimental quality
The evaluation of experimental quality performed following SYRCLE's risk of bias tool showed that studies quality was not sufficient in the majority of studies using omic approaches.Indeed, many criteria were insufficiently reported or lacking such as suitable allocation concealment, random housing, blinding caregivers or investigators and incomplete outcome.Experimental quality was not linked to the type of omic approaches.Improving experimental quality will be important in the future to allow a proper comparison of animal studies and to generalise their findings to clinical studies in humans.

Species, sex and age of rodent models
The majority of animals used to study the effects of cSiO 2 through omic methods were males.While transcriptomics, proteomics, genomics and single-cell omics were performed in males, only transcriptomic techniques were explored in female models.This focus on males in omic studies is relevant for human health as men are more represented among workers exposed to cSiO 2 [1].However, cSiO 2 is also known to induce systemic autoimmunity which is over represented in women [61].Using both male and female models in omic approaches is important considering the potential differential effects of cSiO 2 on both genders [62].As cSiO 2 is able to promote autoimmunity [6], more studies should evaluate the effects of cSiO 2 through omic approaches on mouse strains prone to autoimmunity to better understand the mechanisms involved in cSiO 2 -related autoimmune diseases.The majority of experiments included young animals with a maximal age of sixteen weeks at the beginning of experiment.This could be considered as selection and experimental bias, considering that cSiO 2 effects on oldage models could especially be relevant as a role of senescent cells has recently been suggested in silica-induced pulmonary fibrosis [63].

Types of crystalline silica
Quartz is the most abundant form of cSiO 2 in nature and is used as a raw material in several industrial and building processes.Several quartzes were used in experimental studies such as Min-U-Sil or DQ12.Min-U-Sil 5 is composed of 99% of quartz while DQ12 is a quartz sand composed of 87% of cSiO 2 and 13% of amorphous silica and kaolinite, their particle size is less than 5 µm [60].A large number of studies included in this SLR exposed mice and rats to Min-U-Sil5 or to other types of silica with the same characteristics (Sigma Aldrich, Forsman Scientific).However, the global effects of exposure to artificial stone, containing silica and resins, remains to be explored whereas an epidemic of silicosis was recently observed in artificial stone producer countries [64].

Dosage and frequency of cSiO 2 exposure
Both acute and chronic exposure to cSiO 2 were evaluated through omic techniques in rats and mice.Chronic exposure is relevant to translate the effects of cSiO 2 in mice and rats to human workers but acute exposure can be representative of acute silica hazards such as silicoproteinosis [65].Based on the limitation established for workers exposure, it is determined that 8.28 mg of inhaled cSiO 2 in mice corresponds to an exposure of human workers during 40 years [4].Among acute exposure, the most commonly used dosages in omic studies were less than 5 mg per exposure for mice corresponding to less than one half of human lifetime exposure.Dosage of cSiO 2 for rat acute exposure were mostly higher than 21 mg, which is consistent with the higher weight of the rats.Chronic exposures carried-out in most omic studies consisted in exposing rats to 2 to 15 mg/m 3 of cSiO 2 6 h per day during several days or weeks that is more representative of human workers chronic exposure.Transcriptomic and proteomic techniques were well distributed in the different dosage and frequency of exposure.
Timing of outcome assessment after cSiO 2 exposure is crucial to identify relevant effects.Outcome measurements in omic studies mainly concerned sub-acute effects of cSiO 2 but chronic effects were also well studied with transcriptomic and proteomic approaches.Silicosis is induced by cSiO 2 exposure and is characterised by chronic inflammation and fibrosis [66].As the onset of symptoms classically occurs at a later stage of the disease, sub-acute and long-term effects of cSiO 2 are especially relevant to study such late outcomes.Moreover, longterm effects allow the exploration of autoimmune features such as cSiO 2 -related autoantibody production [13].

Omic methods
Omic approaches such as genomics, transcriptomics, metabolomics or proteomics provide a global perspective from large dataset in organisms and contribute to the understanding of mechanisms involved in human diseases.There are various technologies to study omics but, separately, they cannot explore the entire complexity of organisms and each of these techniques has their strengths and limitations.
Transcriptomic studies were the most omic approaches used in included studies and microarray analysis was more used than RNA-seq.Microarray allows the identification of differentially expressed genes using chip with thousands of short single-stranded DNA sequences, therefore there is an a priori knowledge of the sequences and their number is limited.On the other hand, RNAseq allows sequencing whole transcript of cells or tissues without quantitative limitations and a priori.Studies using RNA-seq were published after 2018 while those using microarray were published earlier corresponding to the development and wider use of RNA-seq characterised by a higher sensitivity and specificity as well as broader data sets with higher comprehensiveness.
Although differentially expressed genes allows a better understanding of the mechanisms involved, it is essential to focus on the proteins encoded by these differentially expressed mRNAs as proteins are the actual actors of the considered physiological and pathological processes.Despite such important considerations, only less than a half of transcriptomic studies validated their results at the protein level.Moreover, proteomic approaches were not widely used in included studies and only a few studies used mass spectrometry techniques and microarrays to identify differentially expressed proteins.Mass spectrometry enables identification and quantification of whole differentially expressed proteins without targeted strategy while protein microarrays require target proteins.Autoantibodies microarray development could be of great interest to profile circulating autoantibodies in autoimmune diseases [67] and, systematically apply such technics in studies assessing the effects of cSiO 2 on adaptive immunity may help decipher the key processes involved in cSiO 2 -related autoimmunity.
Transcriptomics and proteomics provide data on differentially expressed genes and proteins respectively but do not provide direct results regarding cells involved in the process at stake.For this reason, single-cell omics have been developed and single-cell transcriptomics (single-cell RNA-seq), proteomics (mass cytometry CyTOF) or spatial omics (Hyperion) are increasingly used [68].However, single-cell techniques were rarely found in included studies, only one study published in 2021 used single-cell RNA-seq [25].We may expect that the high dimensional single cell analysis at the protein level might be more used to identify which cell types can drive cSiO 2 effects.Multiomic approaches combining transcriptomics, proteomics and metabolomics in a same study and/or protocol may also help improve the overall understanding of the mechanisms involved in the physiopathology of cSiO 2 and foster the design of new therapeutic targets [14].
In included studies, omic approaches were mainly carried out on lungs and more rarely on blood, plasma, serum, spleen and kidney.However this focus on lungs may lead to a gap of knowledge regarding the systemic effects of silica dust [69].Biological processes, pathways, networks and mRNA expression were the most studied omic outcomes in response to cSiO 2 exposure in rodents.Among them, immunity-and inflammationrelated outcomes were the two most frequent domains reported both at the lung and systemic level, suggesting a similar and global response to cSiO 2 exposure.Indeed, a wide range of cytokines were retrieved in response to cSiO 2 .The identification of IL-1β is consistent with the well-described inflammasome (NLRP3) activation induced in response to cSiO 2 , along with the involvement of TLR pathway resulting in IL-1β cytokine release [70].Moreover, the cSiO 2 -induced IL-17 pathways identified in included studies was previously reported [71].This interleukin is known to be increase in autoimmune disorders [72] and could be implicated in cSiO 2 -induced autoimmunity [13].Innate and adaptive immunity both play a role in the response to cSiO 2 exposure.Indeed, terms related to cell activation, functions and recruitment were mainly found at pulmonary and systemic levels, which is consistent with the known recruitment and activation of inflammatory cells in response to cSiO 2 .Phagocytosis were also retrieved in lungs, consistently with existing data regarding the response to cSiO 2 exposure [9].Identification of the complement term was in accordance with its role in silicosis and inflammation [73].The adaptive immunity-related terms such as antibody and T-and B-cell function were especially identified as long-term effects and at systemic levels, that is in coherence with the time of an autoimmune response to cSiO 2 [13,17].All these results from omics studies on rodents enable us to appreciate the overall effects of cSiO 2 exposure in rodents, which are now well described in the literature.Therefore, it would be interesting to investigate the mechanisms involved in its effects and to carry out translational studies to gain a better understanding of the effects of crystalline silica on human pathologies.

Translation of omic results from rodents to humans
Validating results obtained in murine models into humans is an important step in determining whether the results from animal studies are relevant to patients.Among included studies, less than 20% validated in humans the omic results obtained in rodents.More translational studies are thus needed.Indeed, rodent models are required to explore the effects of cSiO 2 since doses and frequencies of exposure can be controlled, making it easier to obtain reproducible and comparable results among studies.However, confirming these results in humans by studying organs, peripheral blood mononuclear cells or serum is still mandatory.Regarding systemic effects of cSiO 2 , none of the included studied validate their results in human.This could be explained by the lack of available human samples, suggesting that fostering the implementation of biorepositories (PBMC, serum and/or plasma biobank) is an important unmet need.Beyond biorepositories, public transcriptome data in humans are available through open access and could enable in silico comparisons with animal results.
Validated results in patients with silicosis or IPF revealed changes in lipid metabolism in response to cSiO 2 exposure in two independent studies [22,24].The AA pathway was found reprogrammed, with an up-regulation of PGD2 and TXA2, two inflammatory mediators potentially involved in silicosis-related fibrogenesis.Such effects may rely on Srebf1, a transcription factor involved in the regulation of AA pathways, that was found downregulated in one of the included study [74,75].Moreover, transcriptome network analyses shown that Srebf1 was connected with some pro-fibrotic gene such as Grem-lin1 [24], that was shown upregulated in two independent studies first in cSiO 2 -exposed mice (long-term effect) and then in lung of patients with IPF [45,56].The overexpression of this protein, as an antagonist of BMP, can imbalance the BMP and TGF-ꞵ pathways, leading to fibrosis [76].Gremlin1 was also upregulated in response to asbestos in the lungs of exposed-mice and to coal dust inhalation in the serum of patient with coal worker's pneumoconiosis [77,78].It was also retrieved at high levels in the serum of SSc patients with interstitial lung disease (ILD), an autoimmune disease for which exposure to cSiO 2 is a risk factor [79].Therefore, Gremlin1 could be use as biomarker of asbestos, coal or cSiO 2 dust exposure.

Perspectives for omic methods to study cSiO 2 effects
This comprehensive overview on cSiO 2 effects highlighted gaps in the literature.1/ cSiO 2 effects on male mouse or rat models are well studied through omic approaches but analysis on female models only focused on transcriptomic approaches and need to be extended.2/ Min-U-Sil silica is more commonly cSiO 2 type used in omic studies.The comparison between Min-U-sil, DQ12 or other type of cSiO 2 exposure through omic methods may help identify key differences in the biological impact of different types of silica.3/ Transcriptomic studies need to be better validated at protein level and/ or in tissues, since proteins are the actual players of the biological response.4/ Systemic effects of cSiO 2 should be further studied and omic techniques should be performed on other samples than lungs such as whole blood, kidney or spleen; notably to explore the autoimmune effects of silica [17].Moreover, only a few omic analyses have been performed on autoimmune disease prone models exposed to cSiO 2 despite the lack of understanding of cSiO 2 -induced autoimmunity.5/ The use of singlecell transcriptomic and proteomic analyses that provide data on each cell type should also be fostered as they allow the identification of specific cell types involved in cSiO 2 -related pathological processes.6/ Among included studies using omic approaches, only a few studies have compared omic results from rodents to omic data from human cohorts exposed to cSiO 2 although such translational approaches are crucial to confirm that biological processes, pathways or networks identified in rodents are also relevant in humans.Translational studies are therefore still needed.

Strengths and limitations
Strengths of the proposal: This SLR is the first study providing a comprehensive and systematic overview of studies exploring the effects of inhaled cSiO 2 in the mouse or rat models using omic approaches.Our SLR follows the PRISMA recommendations for SLRs [18].We used three different databases for article selection providing a comprehensive analysis of the literature on the subject.Moreover, the protocol of this systematic review was published on Prospero prior to the beginning of abstract screening.Combining transcriptomic and proteomic results provide an unprecedented overview of the effects of inhaled cSiO 2 in mouse and rat models.This review includes studies using different time points of outcome measurement after the last cSiO 2 exposure, at both pulmonary and systemic levels.
Limitation of the proposal: we used an a priori definition of omic approaches that is not endorsed or validated.The heterogeneity of the protocols used in the studies (dosage, frequency, duration, cSiO 2 type and methods of exposure) can be considered a limitation as it may preclude a direct comparison of the obtained results.Results may also vary depending on the omic techniques, the platform where the omic approaches are performed and statistical analyses of the data.In addition, we did not specifically explore the impact of the dose or frequency of cSiO 2 exposure.In our work, we only explored the main biological processes, pathways and networks identified in the studies, some pathways are therefore not retained although they may have a role in cSiO 2 effects.By focusing on studies using omic techniques, this review may not include results obtained from others techniques and therefore all the mechanisms modulated by cSiO 2 could not be identified although such aim was beyond the scope of this study.

Conclusion
In this SLR review, providing an overview of cSiO 2 effects in mice and rats, omic techniques were more commonly carried out on lungs and analysis of the systemic effects of cSiO 2 were neglected.Perform further omic analysis on autoimmune prone mouse models and on female models may help identify mechanisms involved in cSiO 2 -induced autoimmunity.Proteomics and single-cell analysis are still lacking to identify the main actors of the pathological processes induced by cSiO 2 and validation in humans are only performed in less than 20% of the available studies.Current validated results from independent omic studies in rodent translated to humans, supported the impact of cSiO 2 on lipid metabolism (AA pathways) and the role of Gremlin1 as a TGF-ꞵ regulator in cSiO 2 -related fibrogenesis.Identifying and validating new prominent pathways could help design and evaluate relevant therapeutic approaches for lung and systemic effects of cSiO 2 .

Fig. 2 Fig. 3 Fig. 4
Fig. 2 Crystalline silica exposure outcomes studied in rat and mouse lungs at several times

Table 2
Characteristics of animals used in omic approaches

Table 3
Characteristics of crystalline silica and exposure

Table 4
Types of omic methods used

Table 5
Validation of transcriptomic results at the protein level

Table 6
Validation of results from mouse and rat models in humans